Cold cream combination of Garcinia mangostana L. Anredera cordifolia (Ten.) and Centella asiatica extracts on Burn Healing Activity Test
Dewa Ayu Swastini1*, I Nyoman Krisna Udayana1, Cokorda Istri Sri Arisanti2
1Laboratory of Pharmacology and Clinical Pharmacy, Department of Pharmacy,
Faculty of Mathematic and Sciences, Udayana University, Bali, Indonesia 80361.
2Laboratory of Technology Pharmacy, Department of Pharmacy,
Faculty of Mathematic and Sciences, Udayana University, Bali, Indonesia 80361.
*Corresponding Author E-mail: ayu_swastini@unud.ac.id
ABSTRACT:
Background: Mangosteen peel (Garcinia mangostana)L., binahong leaf (Anredera cordifolia (Ten.)), and centella herb (Centella asiatica) have been shown to have activity in each phase of burn healing process. Formulating these three plants extract into cold cream preparations is expected to increase the healing activity of burns. Objective: This study aimed to prove the efficacy of the cold cream combination of these plants by examining in vivo burn healing activity using a deep second-degree burn rat model. Materials and Methods: Rat were divided into nine experimental groups (normal, saline solution, silver sulfadiazine, cold cream base, each extract, combination of three extracts, and coldcream combination of three extracts). The burn activity was assessed by inflammatory cell, agiogenesis and collagen formation through histopathological examination. Results: Cold cream combination of mangosteen peel, binahong leaf, and centella herb enhanced the burn wound healing as indicated by progressive improvement in wound healing during 21 days after treatment. Histological result showed that coldcream combination group can reduced inflammatory cell infiltration and increased collagen significantly (p ≤ 0.05), in the other hand same as normal control, but has no effect on angiogenesis. Conclusion: Based on these results, cold cream combination of mangosteen peel, binahong leaf, and centella herb enhanced has a potential effect as an burn healing agent.
KEYWORDS: Burn healing, Cold cream, Mangosteen peel, Binahong leaf, Centella herb.
INTRODUCTION:
Xanton derivatives contained in mangosteen peel are known to have antioxidant and antimicrobial activity that play a role in the inflammatory process4,5.
The saponin in binahong leaves are thought to be able to accelerate the process of wound closure and epithelization through its ability to produce collagen factor I6. Hypertropic scars and keloids that results of wound healing are also seen frequently after burn wounds7. Ethanol extract from centella asiatica can prevent the formation of hypertrophic scars and keloids due to burns7. The combination of the three plants extract above are expected to work synergistically in all four phases of burn healing process. Cold cream is an emulsion of water in oil which has the characteristic of giving a cold effect to the skin. The outer phase of cold cream in the oil form, causing cold cream to be attached longer to the skin so that it is expected to provide a longer therapeutic effect8,9. This study was conducted to evaluate the effect of cold cream extract combination in vivo burn healing activity on a deep second-degree burn injuries in rats.
Table 1. Cold cream formula of mangosteen peel, binahong leaves, and centella herbs extract
|
Formula |
Ingridient |
Amount (gram) |
||||
|
I |
II |
III |
IV |
V |
||
|
Ethanol extract of Mangosteen peel |
Active compounds |
- |
10 |
10 |
- |
- |
|
Ethanol extract of binahong leaves |
Active compounds |
- |
10 |
- |
10 |
- |
|
Ethanol extract of centella herb |
Active compounds |
- |
1 |
- |
- |
1 |
|
Cera alba* |
a/m; stiffening agent |
12 |
12 |
12 |
12 |
12 |
|
Cetaceum* |
Oily phase base; Emolien |
12,5 |
12,5 |
12,5 |
12,5 |
12,5 |
|
Liquid Paraffin * |
Oily phase base; Emolien |
56 |
56 |
56 |
56 |
56 |
|
Natrium tetraborate* |
Preservative |
0,5 |
0,5 |
0,5 |
0,5 |
0,5 |
|
Distilled water * |
Watery phase base; Emolien |
19 |
19 |
19 |
19 |
19 |
MATERIALS AND METHODS:
Plants Material Extraction:
Mangosteen peel extract:
Fresh mangosteen peels were washed with clean water, cut and chopped first, and weighed as much as 100 grams. Followed by the maceration process for 24 hours with 100mL of ethanol 95%. The extracts were evaporated in an oven at a temperature of 45-50°C until it had a volume of less than 10mL. The extract was then cooled to room temperature10.
Binahong leaf and Centella extract:
Binahong leaves and centella herba powder were extracted using the maceration method for one day. The ratio between the leaves and ethanol 70% was 1: 10. The macerate separated from the residue. Repeat the residue maceration two times with the same solvents. All macerates were collected and evaporated at 68°C11.
Cold Cream Formulations:
The oil phase (cera alba, cetaceum, liquid paraffin) and the water phase (distilled water) are heated in a separate container in a water bath at 70°C until they melt completely. The water phase is entered into the oil phase little by little and is stirred constantly using a magnetic stirrer at 400 rpm until a creamy period is formed. Then each extract is added to the cream period which has formed at the same temperature with constant stirring12. The formulation displayed in table 1.
Burn Healing Activity Test:
Fourty-five male Wistar white rats (200-300 gram) were randomly divided into nine groups (normal control, negative control, positive controls (silver sulfadiazine), three combination extracts group, cold cream basis group (I), cold cream with three combination extracts (II), mangosteen extract cold cream (III), binahong extract cold cream (IV), and centella extract cold cream (V). Acclimatization was performed for one week before the experiment.
Burn Wound Model:
The rats were anesthetized by intraperitonial of ketamin (90mg/kg). After shaving their backs hair by blade, a deep second degree burn wound created by a silinder metal with diameter 1,5 cm, weight 50 gram that was heated to 75°C for 10 seconds. The animal were resuscitated with an intraperitonial injection of 5 ml normal saline solution. Thereafter, treatment was initiated for the groups13.
Histopathology Scoring:
Skin tissue samples were taken for histological studies on day 21st with a small excision containing part of the wound area. Tissues were fixed in 10% formalin. Paraffin-embedded sections (5-μm thick) were prepared and stained with hematoxylin and eosin. Light microscopy was used to evaluate the scoring of pathological parameter changes, such as inflammation, angiogenesis and formation of collagen. The parameter was evaluated in 5 different fields (x400) using an ocular microscope and then the averaged of this number was evaluated according to Table 214. All data were analyzed using statistical software SPSS (version 20.0).
Table 2. Histopathology Healings (Scoring)
|
S. No |
Criteria |
Score |
Observation |
|
1 |
IInflammatory Cells Infiltration |
+1 |
Inflammatory cells spread with low density (1-50 cell per field of view) |
|
+2 |
Inflammatory cells spread with medium density (>50 -100 cell per field of view) |
||
|
+3 |
Inflammatory cells spread with tight density (>100-200 cell per field of view) |
||
|
+4 |
Inflammatory cells spread with very tight density (>200 cell per field of view) |
||
|
2 |
Angiogenesis |
+1 |
Capillaries in the wound area spread with low density (1-20 capillary / field of view) |
|
+2 |
Capillaries in the wound area spread with medium density (>20-50 capillary / field of view) |
||
|
+3 |
Capillaries in the wound area spread with tight density (>50-75 capillary / field of view) |
||
|
+4 |
Capillaries in the wound area spread with very tight density (>75 capillary / field of view) |
||
|
3 |
Collagen formation |
+1 |
The density of collagen fibers in the wound area is low |
|
+2 |
The density of collagen fibers in the wound area is medium |
||
|
+3 |
The density of collagen fibers in the wound area is tight |
||
|
+4 |
The density of collagen fibers in the wound area is very tight |
Data obtained in all groups were compared with each other using the parametric ANOVA test with a confidence level of 95% and continued with Bonferroni's post hoc test for multiple comparisons The ethical approval of this study was issued by The Ethical Committee of the Faculty of Veterinary Medicine Udayana University number 195/KE-PH-Lit-1/VIII .
RESULTS:
Wound healing involves a cascade of events characterized by completion of biological processes in certain order and time frame. Histological finding indicated a progressive improvement in wound healing during 21 days after starting treatment. The wound healing parameters that observed included inflammatory cell infiltration, angiogenesis, and collagen formation, which represents each phase of wound healing (figure 1).
The highest number of inflammatory cells was found in the negative group and centella cold cream, and the lowest one found in combination cold cream (p<0.05). Statistically shows that all treatment groups have significant differences in angiogenesis only with normal controls. The cold cream combination had the highest collagen density level followed by binahong and mangoosteen cream, and had significantly different collagen density (P <0.05) when compared to the other groups. The result also shows that this three of the treatment groups had collagen density the same as normal skin conditions (p<0.05) (figure 2).
Figure 1 : Microscopic view of burned area at 21th day after treatment (HandE, x400). Combination Cold Cream (d) has a histology that approaches the normal group (a)
Figure 2. Histopatological Scoring. (a) significantly different from negative gr. (*) significantly different from normal grup.
DISCUSSION:
Our result showed that combination of three extracts in cold cream was efficacious in treatment of second degrees burn when it compared with extract combination and all single extract of cold cream. The wound healing mechanism of a combination cold cream is to reduce inflammation cell infiltration and increase collagen density.
Flavonoids and tannins that contained in mangosteen peel and binahong leaf was known to have anti inflammatory activity15,16,17,24. Flavonoid can bind the bacterial proteins so that it can disrupt the metabolism of microorganisms4. While tannin compounds work as an antibacterial by coagulating the protoplasm of germs4. Saponin that contained in the three extracts has antibacterial activity that works by interfering with the stability of bacterial cell membranes, thus causing bacterial cell lysis18. Formulated extracts in coldcream increased the humidity, made damage tissue became decay, its will stimulated neutrophil cell to phagocytosis the tissue and decreased the inflammation time19.
Saponin compounds in three extracts are thought to accelerate the formation of new blood vessels in the healing process through Vascular Endhotelial Growth Factor20. Moist conditions in wounded skin can also increase angiogenesis21. The application of cold cream provides a moist condition on the wound so it will be trigger the mobilization of macrophages and activate TNF-cells to secrete growth hormone or chemotatic factors of blood vessel endothelial cells, that play a role in the process of angiogenesis22.
In injured skin, Reactive Oxygen Species (ROS) are produced when neutrophils degrade bacteria and other foreign bodies in the process of phagocytosis23. ROS can cause newly formed collagen to degrade, and also attack lipids on the skin resulting in lipid peroxidation which causes damage and cell death in the injured area. The antioxidant activity possessed by flavonoids and xanton contained in mangosteen peel and binahong leaf extract is known to be effective in reducing ROS levels so that newly formed collagen can proliferate normally23.
The saponin in the binahong leaf is able to accelerate the process of wound closure and epithelialization through its ability to produce collagen factors I6,24. While asiaticoside contained in the centella can facilitate wound healing by increasing the excitability and increasing collagen synthesis7. Cold cream preparations can provide a moist atmosphere to the skin, its will induced fibroblast proliferation and collagen synthesis increase up to 50%21.
From histopathological observations, it can be seen the cold cream combination have a synergistic effect in each phase of wound healing, this activity became more optimal when this extract combination were formulated in coldcream preparation
ACKNOWLEDGEMENTS:
Departement of Research and Community Services, Udayana University.
CONFLICT OF INTEREST:
The authors declare no conflict of interest
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Received on 06.05.2020 Modified on 27.06.2020
Accepted on 25.07.2020 © RJPT All right reserved
Research J. Pharm. and Tech. 2021; 14(5):2483-2486.
DOI: 10.52711/0974-360X.2021.00437